Validation of the luminescent bacteria test

Status:

completed 11/2003

Aims:

Workplace air is often contaminated by a number of chemical substances. Chemical analyses may be performed for detection of the individual substances. They do not however yield any indication of the overall toxicity of the gaseous or vaporous substances in the atmosphere. The luminescent bacteria test may represent a convenient method for assessment of the overall toxicity of complex mixtures (indicator principle in accordance with technical rule TRGS 403 governing the assessment of substance mixtures in workplace atmospheres). This is particularly the case when the mixtures contain substances for which atmospheric limit values do not exist, or unknown or little-studied substances, i. e. substances which cannot be assessed by conventional methods. A method has already been developed in an earlier project (BIA7003 concerning thermodesorption for the preparation of samples for bacterial toxicity tests) by which complex gaseous substance mixtures can be sampled and exploited for an aqueous toxicity and mutagenicity test. The parameters of the method were now to be optimized and its applicability tested on a greater number of substance groups. At the same time, the project was to demonstrate that the method is suitable for concentrations in the MAK (maximum workplace concentration) ranges for airborne pollutants, and that the luminescent bacteria test employed (Microtox test) is sufficiently sensitive in these concentration ranges. The long-term objective was the development of a validated BIA measurement method.

Activities/Methods:

Test gas concentrations in the MAK value range of the individual substances were manufactured in the test gas apparatus of the BG Institute for Occupational Safety (BIA) and tested by means of air toxic tubes (initially single substances only). The sampling tubes were desorbed by thermodesorption employing cryofocussing, and the gases obtained collected in small volumes of ethanol. The method was optimized by adjustment of the desorption parameters on the thermal desorber, and the recoveries of the ethanol solutions detected by means of gas chromatography/electron capture detector (GC-ECD). The toxicity of aliquots of the ethanol samples obtained with the optimum parameters was tested and the sensitivity of the luminescent bacteria (species: Vibrio fischeri) determined.

Results:

A method developed by the BIA for the supply of defined test gases to air toxic tubes, subsequent thermodesorption, and the DIN-certified luminescent bacteria screening test involving test substances, had not been studied adequately with regard to reproducibility and experimental conditions suitable for general application of the method to a large number of chemical compounds and mixtures of substances. In a study of the ideal process conditions for thermodesorption, the recovery rates of 2-butanone, ethyl acetate, n-hexane, n-nonane, n-octane, tetrachloroethane, toluene and xylene were measured experimentally as a function of various measurement parameters (carrier gas flow rates in the test gas stream, thermodesorption flow rates, supply to the detector tubes in different concentrations and at different flow rates) and validated. Recovery rates > 80% in the MAK value range were measured analytically in the great majority of cases for the compounds studied. The parameters of the method were standardized in order to include a wide spectrum of dangerous substances in conjunction with a high yield. Relevant dangerous substances for thermodesorption and the Microtox luminescent bacteria test were selected from the following classes of compounds: aromatic, cyclic, aliphatic hydrocarbons, ester, aldehydes, alkyl alcohols, chlorinated compounds, ether, ketones, phthalates, organotin compounds and isocyanates. The luminescent bacteria test returns clearly positive inhibition values in the MAK value range for the majority of the selected compounds in ethanol test solutions and in test gases. The combination of thermodesorption and Microtox test represents a proven laboratory test method for discrete substances. In subsequent project stages, mixtures of harmful substances are to be tested separately, in products, and in samples taken at actual workplaces in the course of in-plant measurement procedures.

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